Process for the production of a saccharase inhibitor

ABSTRACT

The invention relates to a saccharase inhibitor derived from Actinoplanaceae Strain CBS 961.70, including mutants and variants thereof, means for the production of said saccharase inhibitors comprising cultivation of Actinoplanaceae Strain CBS 961.70, including mutants and variants thereof, in appropriate nutrient solutions which are characterized by being starch free under conditions most favorable to growth and production of said saccharase inhibitor and recovering a saccharase inhibitor from culture broths of said nutrient solutions, as well as the use of said inhibitor in pharmaceutically acceptable therapeutic compositions suitable for use in the treatment and relief of conditions indicative of adiposity, diabetes, pre-diabetes, hyperlipaemia (atherosclerosis), caries and the like.

BACKGROUND OF THE INVENTION

It is known that in animals and man, after consumption of foodstuffs andbeverages containing saccharose, hyperglycaemias occur which are causedby rapid splitting of the saccharose by saccharases of the digestivetract according to the following equation ##STR1## These hyperglycaemiasare particularly strong, and of long-lasting pronounced character, withdiabetics. With adipose subjects, the alimentary hyperglycaemiafrequently causes a particularly intense insulin secretion which in turnleads to increased fat synthesis and reduced fat degradation.

These saccharase inhibitors according to the invention, obtained andisolated according to the above methods, considerably reduce thealimentary hyperglycaemia and hyperinsulinaemia in rats dose withsaccharose.

It is furthermore known that caries occur particularly strongly andfrequently after consumption of beverages and foodstuffs containingsaccharose [for example, B. W. Gold: Advances in Applied Microbiology 11(1969) 135-157]. An inhibition of the splitting of saccharose by theinhibitors according to the invention reduces the formation ofcariogenic substances.

These inhibitors are therefore suitable for use as a therapeutic agentfor the following indications: adiposity, hyperlipaemia(atherosclerosis), diabetes, pre-diabetes and caries.

RELATIONSHIP TO COPENDING APPLICATIONS

U.S. patent application Ser. No. 213,066, filed Dec. 28, 1971, entitled"GLYCOSIDE-HYDROLASE ENZYME INHIBITORS" discloses that microorganisms ofthe order Actinomycetales form inhibitors for glycoside-hydrolases, andin particular inhibitors for glycoside-hydrolases of preferentiallycarbohydrate-splitting enzymes of the digestive tract. One group ofthese inhibitors is relatively heat-stable and stable to acid and alkaliat room temperature. Chemically speaking, these inhibitors belong to theclass of the oligosaccharides or polysaccharides or their derivatives.

Thus, for example, in Examples 28-38 of the aforesaid applicationdescribes the isolation of such an amylase inhibitor derived from theActinoplanaceae strain CBS 961.70 belonging to the orderActinomycetales. The culture solutions resulting from fermentations ofthis strain under optional conditions contain about 30,000-40,000AIU/ml., and the purest inhibitors isolated therefrom possess specificactivities of 3-8 × 10⁶ amylase inhibitor units (AIU μ g).

In copending application Ser. No. 336,687, filed Feb. 28, 1973 there isdisclosed the production of amylase inhibitors from strain CBS 614.71 bycultivating the same in a nutrient medium containing starch.

THE PRESENT INVENTION

It has now been discovered that by the use of the Actinoplanaceae strainCBS 961.70, including the new strain CBS 614.71 and its mutants andvariants, in conjunction with nutrient solutions which are free fromstarch cultures containing more than 10 SIU/ml can be obtained.

The invention, accordingly, provides a novel process for the productionof a saccharase inhibitor which comprises culturing a microorganism ofthe family Actinoplanaceae, Strain CBS 961.70, a mutant or variantthereof, in a nutrient culture which does not contain starch underaerobic conditions for a period of time sufficient for the production ofsaid saccharase inhibitor and separating said saccharase inhibitor fromsaid nutrient culture medium.

As used herein, the expression "a mutant or variant" of Strain CBS961.70 is defined as strains derived from Strain CBS 961.70 by mutationseither spontaneous or induced such as by bombardment with mutagenicradiation and variants of the strain produced from it by processes otherthan mutations. Strain CBS 614.71 is an example of such a mutant orvariant.

In the specification and claims the abbreviation "CBS" refers to strainsthat have been deposited at the Centraalbureau voor Schimmelcultures,Baarn, Netherlands, under the stated Accession Number.

It has been found that the culture solutions of the Strain CBS 961.70prepared in accordance with the techniques of copending application Ser.No. 213,066, filed Dec. 28, 1971, frequently also possess a slightsaccharase-inhibiting activity, but the isolation and preparation inpure form of the saccharase inhibitor formed is very difficult due tothe presence of considerable amounts of amylase inhibitor in the culturesolution.

It has now been discovered that it is possible to suppress the formationof amylase inhibitor while at the same time promoting the formation ofsaccharase inhibitor.

This invention provides a method of producing a saccharase inhibitorcomprising growing Actinoplanaceae Strain CBS 961.70 or a mutant orvariant thereof in a starch-free nutrient medium to produce a culture,and separating the saccharase inhibitor from the culture.

In this method, the nutrient medium must be free of starch. A preferrednutrient medium is a solution having the composition 3.5% of glucose,0.5% of casein hydrolysate, 1.3% of yeast extract, 0.3% of K₂ HPO₄ and0.3% of CaCO₃ ; pH adjusted to 7.8 with KOH before sterilization. Suchsolutions after innoculation and several days' fermentation with StrainCBS 961.70, yield cultures which contain more than 10 SIU/ml (SIU =Saccharase Inhibitor Unit). At a content of 300-700 AIU/ml (AIU =Amylase Inhibitor Unit) an SIU/AIU ratio of 15 × 10.sup.⁻³ -30 ×10.sup.⁻³ results. (f = SIU/AIU × 10³).

The yield of saccharase inhibitor in the method of the invention can beincreased further if maltose is added to the nutrient solution. If, forexample, 1.5% of maltose is added to the above-mentioned nutrientsolution, culture broths containing 16-18, and even up to 24, SIU/ml areobtained.

A decisive factor in obtaining a favorable SIU/AIU ratio is the durationof growth. If growth is stopped shortly before reaching, or just onreaching, the maximum SIU content, which depending on the inoculationcan happen after only 1.5 to 2 days' fermentation, f-values of up to 180are obtained.

As has heretofore been stated, instead of the Strain CBS 961.70 it isalso possible to use its mutants and variants such as Strain CBS 614.71.

The saccharase inhibitor can be isolated from the appropriatelyfermented culture solution by any suitable method. One such method isconcentrating the culture broth in vacuo to 1/10-1/20 of the originalvolume, with subsequent lyophilization. Alternatively the inhibitor maybe precipitated from the concentrated broth with 4-10 parts by volume oforganic solvent such as an alcohol or ketone, preferably with 5-9 partsby volume of acetone. One preferred and particularly simple method isadsorption of the inhibitor from the neutral (preferably pH 5-8) culturesolution on 0.5-4%, preferably 1-2% of active charcoal and subsequentdesorption with an aqueous alcohol or aqueous acetone, especially atacid pH values, preferably with 50% strength aqueous acetone at pH 2--3.The desorbate is subsequently concentrated in vacuo to 1/50-1/200,preferably 1/100, of the initial volume (of the culture) and issubsequently precipitated with organic solvents, preferably acetone. Ingeneral, in order to isolate purer preparations, a preliminaryadsorption of the brown dyestuffs which contaminate the culture solutionis preferably carried out on 1- 2% of active charcoal at acid pH values(pH values of 1-4, preferably 2-3), at which low pH, surprisingly, theadsorption of the saccharase inhibitor does not occur to a significantextent.

The present invention also includes a pharmaceutical compositioncontaining as an active ingredient the saccharase inhibitor produced bythe method of this invention, in admixture with a solid or semi-liquiddiluent.

Examples of such diluents include:

(a) fillers and extenders, e.g. starch, sugars, mannitol, and silicicacid; (b) binding agents, e.g. carboxymethyl cullulose and othercellulose derivatives, alginates, gelatine and polyvinyl pyrrolidone;(c) moisturizing agents, e.g. glycerol; (d) disintegrating agents, e.g.agar-agar, calcium carbonate and sodium bicarbonate; (e) agents forretarding dissolution e.g. paraffin; (f) resorption accelerators, e.g.quaternary ammonium compounds; (g) surface active agents, e.g. cetylalcohol, glycerol monostearate; (h) adsorptive carriers, e.g. kaolin andbentonite; (i) lubricants, e.g. talc, calcium and magnesium stearate andsolid polyethylene glycols.

The inhibitor may also be added to foodstuffs and beverages containingsaccharose.

Particular examples of the pharmaceutical compositions of this inventionare suspensions, granules, chewing gum, toothpaste, mouthwash,foodstuffs and beverages containing the saccharase inhibitor.

The saccharase inhibitor produced by the method of this invention isgenerally to be administered orally in amounts of 100-10000 SIU/kg bodyweight per therapeutic administration, which will be once or severaltimes daily before, during or after meals.

The invention further provides medicaments in dosage unit formcontaining saccharase inhibitor produced by the method of the invention.

The term "medicament in dosage unit form" as used here means physicallydiscrete coherent portions suitable for medical administration, eachcontaining a predetermined individual quantity of the inhibitor, thesaid quantity being such that one portion is required for a singletherapeutic administration in accordance with guidelines set forthabove. Examples of such medicaments in dosage unit form according to theinvention are tablets, dragees, capsules, sticks of chewing gum andampoules containing the inhibitor.

AMYLASE TEST

One amylase inhibitor unit (1 AIU) is defined as the amount of inhibitorwhich inhibits two amylase units to the extent of 50%. One amylase unit(AU) is the amount of enzyme which in one minute, under the testconditions indicated below, splits 1 μ equivalent of glucosidic bonds instarch. The μ equivalent of split bonds are determined colorimetricallywith dinitrosalicylic acid as μ equivalent of reducing sugar formed andare quoted, with the aid of a maltose calibration curve, as μ equivalentof maltose equivalents. To carry out the test, 0.1 ml of amylasesolution (20-22 AU/ml) is mixed with 0-10 μg of inhibitor or 0-20 μg ofthe solution to be tested in 0.4 ml of 0.02 M sodium glycerophosphatebuffer/0.001 M CaCl₂ at pH 6.9 and equilibrated for about 10-20 minutesin a water bath at 35° C. The mixture is then incubated for 5 minutes at35° C with 0.5 ml of a 1% strength starch solution (soluble starch ofMessrs. Merck, Darmstadt, No. 1252) which has been prewarmed to 35° C,and 1 ml of dinitrosalicylic acid reagent (according to P. Bernfeld inColowick-Kaplan, Meth. Enzymol., Volume 1, page 149) is then added. Todevelop the color, the batch is heated for 5 minutes on a boiling waterbath, then cooled and treated with 10 ml of distilled water. Theextinction at 540 nm is measured against a correspondingly made-up blankwithout amylase. For evaluation, the amylase activity which is stillpresent after addition of inhibitor is read off from a previouslyrecorded amylase calibration curve and the percentage inhibition of theamylase employed is calculated therefrom. The percentage inhibition isplotted as a function of the quotient ##STR2##

SACCHARASE TEST

One saccharase inhibitor unit (SIU) is defined as the amount ofinhibitor which inhibits two saccharase units to the extent of 50%. Onesaccharase unit (SU) is the amount of enzyme which in one minute, underthe test conditions indicated below, splits 1 μmol of saccharose intoglucose and fructose. The μmol of glucose formed is determinedquantitatively with the aid of the glucose oxidase reaction underconditions under which further splitting of saccharose by the saccharaseno longer occurs. To carry out the test, 0.05 ml of a saccharasesolution¹) adjusted to 0.12 SU is mixed with 0-20 μg of inhibitor or0-20 μl of the solution to be tested and made up to 0.1 ml with 0.1 Msodium maleate buffer of pH 6.0. The mixture is equilibrated for 10minutes at 35° C and then mixed with 0.1 ml of an 0.05 M saccharosesolution in 0.1 M sodium maleate buffer of pH 6.0, which has beenprewarmed to 35° C. The mixture is incubated for 20 minutes at 35° C andthe saccharase reaction is stopped by adding 1 ml of glucose oxidasereagent²) and incubated for a further 30 minutes at 35° C. Thereafter, 1ml of 50% strength H₂ SO₄ is added and the mixture is measured at 545 nmagainst a corresponding blank. For evaluation, the percentage inhibitionof the saccharase employed is calculated and converted, from the 50%inhibition point, with the aid of a glucose calibration curve, to SIU/gor SIU/liter.

The following examples illustrate the best mode now contemplated forcarrying out the invention.

EXAMPLE 1

1 liter Erlenmeyer flasks containing 120 ml of a nutrient solution ofcomposition 3.5% of glucose, 0.5% of casein hydrolysate, 1.3% of yeastextract, 0.3% of CaCO₃, 0.3% of K₂ HPO₄, (the pH being adjusted prior tosterilization to 7.8 with KOH and the solution being sterilized for 30minutes at 121° C), are inoculated with 4 ml of a 3-day old pre-cultureof Strain CBS 961.70 in a nutrient solution of composition 3% ofglycerine, 3% of soya flour and 0.2% of CaCO₃, obtained by incubation ona rotary shaking machine at 28° C, and the charges are incubated at 24°C after 3 days of incubation, a culture broth is obtained which contains9.1 SIU/ml and 240 AIU/ml and after 4 days' incubation contains 10.4SIU/ml and 675 AIU/ml.

EXAMPLE 2

When maltose in various concentrations is added to the nutrient solutionused in Example 1 and the solution is inoculated and incubated accordingto Example 1, the following yields of SIU/ml and AIU/ml are obtained:

    ______________________________________                                        Maltose                                                                       concentra-                                                                             After 3 days    After 5 days                                         tion %   AIU/ml    SIU/ml    AIU/ml  SIU/ml                                   ______________________________________                                         0       363       11.2      444     8.8                                      0.5      479       12.7      614     11.3                                     1.0      383       13.6      688     12.5                                     1.3      337       15.7      725     12.7                                     1.8      274       16.6      678     14.4                                     2.2      287       16.8      600     15.5                                     ______________________________________                                    

EXAMPLE 3

To the nutrient solution of Example 1 is added 1.3% of maltose and theprocedure of Example 1 is repeated, a culture broth is obtained whichafter 2 days' fermentation contains 14.4 SIU/ml and 81 AIU/ml and after4 days' fermentation contains 14.5 SIU/ml and 580 AIU/ml.

EXAMPLE 4

1 liter Erlenmeyer flasks which contain 120 ml of a nutrient solution ofcomposition 3% of glucose, 1% of maltose, 0.5% of casein hydrolysate,1.3% of yeast extract, 0.3% of CaCO₃, 0.3% of K₂ HPO₄ (the pH beingadjusted to 7.2 with Na₂ CO₃ before sterilization and the mixture beingsterilized for 30 minutes at 121° C) are inoculated with 4 ml of a 3days' old pre-culture of Strain CBS 961.70 in a nutrient solution ofcomposition 3% of glycerine, 3% of soya flour, 0.2% of CaCO₃, obtainedby incubation on a rotary shaking machine at up to 28° C, and the batchis incubated at 24° C after 4 days of incubation, a culture broth isobtained which contains 22 SIU/ml and 500 AIU/ml.

EXAMPLE 5

500 ml of a brown culture solution of the Strain CBS 961.70 fermentedaccording to Example 4 and containing 22,000 SIU/liter were adjusted topH 2.5 with half-concentrated HNO₃, treated with 5 g of active charcoal(Messrs. Merck) and stirred for 10 minutes. The mixture was centrifugedfor 15 minutes at 10,000 rpm and the clear,, yellow supernatant liquidwas neutralized with ammonia. Thereafter it was concentrated to 50 ml ona rotary evaporator at 20 mm Hg and this concentrated solution wastreated with 50 ml of acetone to precipitate inactive constituents. Theclear filtrate was added dropwise to 400 ml of acetone while stirringand the precipitate which formed was collected on a filter, washed withacetone and ether and dried in vacuo at room temperature.

Yield: 1.4 g with 6,000 SIU/g = 70% yield, relative to the activity.

EXAMPLE 6

500 ml of a culture solution of the Strain CBS 961.70 fermentedaccording to Example 4 and containing 22,000 SIU/liter were worked upaccording to Example 5 but were precipitated with ethanol instead ofwith acetone.

Yield: 0.6 g with 7,000 SIU/g = 38% yield, relative to the activity.

EXAMPLE 7

1 liter of a dark brown culture solution (21,000 SIU/liter) fermentedanalogously to Example 4 was adjusted to pH 2.5 with half-concentratednitric acid, 10 g of active charcoal (Messrs Merck) and 10 g of thefilter aid Clarcell were added and the mixture was stirred for 10minutes. It was filtered through a filter provided with a 1-2 cm highClarcell layer and the filtrate was neutralized with ammonia. The filtercake was discarded. 1.5% of active charcoal were added to the neutralfiltrate (19,000 SIU/liter) and the mixture was stirred for 10 minutesand then again filtered. The filtrate still contained approximately 10%of the activity and was discarded. (If this residual activity is also tobe isolated, a further adsorption with 0.5% of active charcoal iscarried out and the charcoal residues are combined). The charcoalresidue containing the saccharase inhibiting activity was washed with alittle water and then stirred 3 times for 10 minutes with 50 ml portionsof 50% strength acetone at pH 2.5 (HCL) for desorption of the activity.Thereafter, the mixture was in each case filtered and the threedesorbates were combined. They were concentrated to 10 ml in a rotaryevaporator and the viscous concentrate was treated with the same volume(10 ml) of methanol, while stirring. The precipitate formed wascentrifuged off. The clear light brown 50% strength solution in methanolwas then added dropwise, while stirring, to 250 ml (= 12.5 volumes) ofabsolute acetone. The precipitate formed settled out well and afterdecanting the deep yellow supernatant liquid the residue was taken up inabsolute acetone and washed. It was filtered off and further rinsed oncewith absolute acetone and once with ether. Thereafter it was dried invacuo at room temperature.

Yield: 850 mg with 12,500 SIU/g = 51% yield (relative to units in theculture broth).

                                      TABLE                                       __________________________________________________________________________    (Activity yields from working up)                                             __________________________________________________________________________                                     ##STR3##     SIU                                            Volume                       Yield                             Working-up Step                                                                              (ml) SIU/L AIU/L ×10.sup.3                                                                      SIU   (%)                              __________________________________________________________________________    1) Original solution                                                                         1,000                                                                              21,000                                                                              0.5×10.sup.6                                                                  42     21,000                                                                             100                               2) After 1st active charcoal                                                     adsorption (pH 2.5)                                                                       1,000                                                                              19,000                                                                              0.4×10.sup.6                                                                  48     19,000                                                                              90                               3) After 2nd active charcoal                                                     adsorption (pH 7.0)                                                                       1,000                                                                              2,000              2,000                                                                              (discard-                                                                     ed: 10%)                          4) 1st desorbate                                                                             45   260,000                                                                             3.5×10.sup.6                                                                  75     11,500                                                                              55                               5) 2nd desobate                                                                              45   92,000                                                                                2×10.sup.6                                                                  46     4,100                                                                               19.5 81                          6) 3rd desobate                                                                              45   30,000                                                                              0.75×10.sup.6                                                                 40     1,350                                                                               6.5                              7) Concentrated to 10 ml on                                                      rotary evaporator                                                                         10   1,500,000                                                                            22×10.sup.6                                                                  68     15,000                                                                              72                               8) 50% methanol precipita-                                                       tion (filtered)                                                                           18   730,000                                                                              12×10.sup.6                                                                  61     13,200                                                                              63                               9) Supernatant liquid from                                                       acetone precipitation                                                                     260  8,400              2,200                                                                              (discard-                                                                     ed: 10%                           10)                                                                              Precipitate 0.85 g                                                                             12,500/g                                                                            0.22 ×10.sup.6 /g                                                             56     10,600                                                                              51                               __________________________________________________________________________

EXAMPLE 8 (Experimental arrangements for demonstrating the action ofsaccharase inhibitors in rats)

To produce an alimentary hyperglycaemia and hyperinsulinaemia fastingrats (n = 6) are given 2.5 g/kg of saccharose orally. Six other rats aregiven, additionally to the saccharose, the saccharase inhibitormanufactured analogously to Example 5, in the indicated dosage. Theblood glucose and the serum insulin are examined at the indicated timeintervals after administration of saccharose. The blood samples areotained from the retroorbital venous plexus. Blood glucose is determinedin an autoanalyzer [Technicon®, according to Hoffman: J. biol. Chem.120, 51 (1937)], and serum insulin according to the methods of Hales andRandle: Biochem. J. 88, 137 (1963).

    __________________________________________________________________________    TABLE 1                                                                       __________________________________________________________________________    Average blood glucose values in mg/100 ml ± ls of fasting rats at          various times after oral                                                      administration of saccharose ± active compound analogously to Example      Dose/kg, administered orally                                                                 10     20     30     60     120 min.                           __________________________________________________________________________    Control without saccharose                                                                   67 ± 7.1                                                                          72 ± 4.6                                                                          68 ± 5.3                                                                          66 ± 5.0                                                                          71 ± 3.3                        Control with saccharose                                                                      134 ± 15                                                                          140 ± 16                                                                          126 ± 12                                                                          117 ± 20                                                                          103 ± 3.0                       100 SIU in saccharose                                                                        96 ± 4.4                                                                          93 ± 6.2                                                                          92 ± 5.6                                                                          92 ± 3.4                                                                          95 ± 12                         Legend: -                                                                                     ##STR4##                                                                             ##STR5##                                                                             ##STR6##                                                                             ##STR7##                                  ##STR8##                                                                 

    TABLE 2                                                                       __________________________________________________________________________    Average serum insulin values in μU/ml ± ls of fasting rats at           various times after oral                                                      administration of saccharose ± active compound analogously to Example      Dose/kg, administered orally                                                                 10     20     30     60     120 min.                           __________________________________________________________________________    Control without saccharose                                                                   8.8 ± 1.8                                                                         9.8 ± 1.4                                                                         10.8 ± 2.3                                                                        10.7 ± 4.6                                                                        8.1 ± 0.7                       Control with saccharose                                                                      22.2 ± 11                                                                         35.3 ± 14                                                                         19.3 ± 5.0                                                                        16.7 ± 5.3                                                                        8.1 ± 1.1                       100 SIU in saccharose                                                                        9.9 ± 4.3                                                                         11.1 ± 3.5                                                                        7.6 ± 1.7                                                                         14.8 ± 3.8                                                                        10.2 ± 2.8                      Legend: -                                                                                     ##STR9##                                                                             ##STR10##                                                                            ##STR11##                                        ##STR12##                                                                    __________________________________________________________________________

What is claimed is:
 1. A method of producing a saccharase inhibitorcomprising growing Actinoplanaceae Strain CBS 961.70 or Strain CBS617.71 in a starch-free nutrient medium to produce a culture andseparating the saccharase inhibitor from the culture.
 2. The method ofclaim 1 in which the medium contains maltose.
 3. The method of claim 2in which the medium contains about 1.5% maltose.
 4. The method of claim1 in which the growth is stopped immediately before or at the time whenthe concentration of saccharase inhibitor in the culture reaches itsmaximum.
 5. The method of claim 1 in which the saccharase inhibitor isisolated by adsorption from the culture on active charcoal atsubstantially neutral pH followed by desorption from the charcoal withan aqueous alcohol or acetone.
 6. Saccharase inhibitor produced by theprocess of claim 1.